Cell surface antigens - identification (Sep/01/2006 )

Hi,I have a novel cell line developed from a crustacean. I would like to know how to identify cell surface antigens in this cell type so that i can use it to understand the cell distributioin in vivo. There are no background studies of this nature in this species or any other closely related crustaceae. So literature search is not very productive. Please gicve me some ideas or protocols. Thanks in advance

-smartsunny-

do you mean cell surface receptors?

-strawberry-

QUOTE (strawberry @ Sep 1 2006, 03:36 AM)

do you mean cell surface receptors?

Yes strwberry. I am trying to identify surface antigens tht could be specific to this cell type.

-smartsunny-

How about do a surface labeling with I125 or biotin-NHS then SDS-PAGE/ expossure with film or blot/ bind with Avidin?

-genehunter-1-

QUOTE (genehunter-1 @ Sep 1 2006, 06:26 AM)

How about do a surface labeling with I125 or biotin-NHS then SDS-PAGE/ expossure with film or blot/ bind with Avidin?

I get ur train of thought genehunnter(Tnx a lot): NHS-biotin can bind to almost any protein, so after labelling intact cells, I do SDS_PAGE, separate proteins; detect bands; then maybe excise them & use to raise antibodies?? IS that what u mean?

One more thing - how can I identify what protein it is without going oin for protein sequencing (too expensive, can't afford it)?

-smartsunny-

Yes, It helps for identifying membrane proteins (Extracellular domain), as such derivcative is not membrane permeable. Also if you use lactoperoxidase and I125, it is also external labeling method. Of course you can isolate these afterwards. Peptide sequencing may not be as expansive as you may think, if you only pick a few major ones. (well I am not sure your funding situation...) There is littler alternative that is effective and cheap for this purpose. Maybe raise your own Ab wouls be one. You can do other neat stuff with them down the road.

-genehunter-1-

-as genehunter said, first u have to label your cells with biotin (biotinylation)-lyse the cells -do affinity chromatography using Avidin supported gel, now u suppose that all biotinylated proteins will bind to avidin molecules of the gel-wash the gel so all non-biotinylated proteins are washed away-elute your gel to free your proteins-and as u said, u can run gel electrophoresis to estimate the MW of the proteins and use them to raise antibodies

one more thing to say: there 'r many labelling methods u can finde.g.u can select the biotinylated proteins with HRP(horse raddish peroxidase) which is bound to Avidin

i don't know methods other than sequencing to identify new proteins...

good luck..

-strawberry-

QUOTE (strawberry @ Sep 1 2006, 11:57 AM)

-as genehunter said, first u have to label your cells with biotin (biotinylation)-lyse the cells -do affinity chromatography using Avidin supported gel, now u suppose that all biotinylated proteins will bind to avidin molecules of the gel-wash the gel so all non-biotinylated proteins are washed away-elute your gel to free your proteins-and as u said, u can run gel electrophoresis to estimate the MW of the proteins and use them to raise antibodies

one more thing to say: there 'r many labelling methods u can finde.g.u can select the biotinylated proteins with HRP(horse raddish peroxidase) which is bound to Avidin

i don't know methods other than sequencing to identify new proteins...

good luck..

Thank you everybody. Now I have to gt back to my supervisor and have a serious talk with him. If I get any good results I'll share it...